JPH04346934A - Liquid preparation of gamma-globulin - Google Patents

Abstract

PURPOSE:To obtain a stable liquid preparation of non-chemical modified complete molecule type gamma-globulin, capable of being administered to a patient of sorbitol dysbolism, having no increase in polymer of gamma-globulin in organism administration as well as in long-term preservation, no rise in anticomplementary value, maintaining excellent appearance and properties during long-term preservation. CONSTITUTION:A liquid preparation capable of being intravenously administered, not showing side effects caused by sorbitol to a patient of sorbitol dysbolism, having excellent storage stability, comprising a solution containing non-chemical modified complete molecule type gamma-globulin and having low conductivity, preferably the solution having >95 % monomer content of non-chemical modified complete molecule type gamma-globulin, having <=1mmho, especially <=0.6mmho conductivity (calculated at 8 deg.C), substantially not containing sorbitol, having about pH 5.5+ or -0.2, preferably pH about 5.5, preferably containing one or more stabilizers selected from polyethylene glycol, human serum albumin and D- mannitol.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention provides a liquid pharmaceutical composition of non-chemically modified complete molecular type γ-globulin that can be injected intravenously and has excellent storage stability, which can also be used for patients with sorbitol metabolic disorders. Regarding.

0002

[Prior Art/Problems to be Solved by the Invention] Among non-chemically modified immune γ-globulins, which are plasma protein components, non-chemically modified γ-globulin preparations containing IgG as a main component have been widely used to treat various infectious diseases. It has been used for prevention and treatment. By the way, γ-globulin, in a solution state, easily becomes a polymer during storage, causing side effects when administered intravenously, so that it has conventionally been formulated in a freeze-dried manner. In addition, non-chemically modified complete molecular type γ-
Considering the instability of globulin in solution state,
Freeze-drying is the best method, and the importance of freeze-drying is well known.

On the other hand, compared to dry preparations, liquid preparations have the advantage of not requiring dissolution in distilled water for injection and can be easily administered, but as mentioned above, their poor stability has hindered their practical use. I was late.

[0004] Recently, a low conductivity solution containing non-chemically modified complete molecular type γ-globulin containing sorbitol has been developed.
An intravenously administrable liquid composition characterized by having a pH of 5±0.2 has also been proposed (Japanese Patent Laid-Open No. 63-19
No. 2724).

However, in patients with sorbitol metabolic disorders, sorbitol-containing preparations may cause serious liver disease.
There are reports that it may cause side effects such as kidney damage [Schul
te, M. J. et al., Lancet, ii, 188, (1
977)], it is dangerous to administer sorbitol-containing preparations to such patients.

[0006] In addition, as a stable liquid preparation, a pH of about 3.
5 to 5, γ with the condition that the ionic strength is less than about 0.001
- A liquid globulin composition has been proposed in JP-A-58-43914.

[0007] However, when such a strongly acidic liquid preparation is administered to a living body, γ-globulin aggregates (polymerizes) in the biological body fluid, which is kept almost neutral, due to the buffering effect of the body fluid itself. This is not necessarily desirable, as there is a risk of it getting lost.

Therefore, the first object of the present invention is to suppress the increase in the amount of γ-globulin polymers both during storage and when administered to living organisms, to prevent an increase in anti-complement value, and to obtain a non-chemically modified product. An object of the present invention is to provide a liquid composition (preparation) of γ-globulin that does not impair the activity of complete molecular type γ-globulin. A second object is to provide a liquid composition (preparation) of γ-globulin that does not exhibit side effects caused by sorbitol even for patients with sorbitol metabolic disorders.

[0009]

[Means for Solving the Problems] In view of the above points, the present inventors have conducted extensive research and have found that the above object is to provide a low conductivity solution containing non-chemically modified complete molecular type γ-globulin, Substantially free of sorbitol, approximately 5.5±0.2
We have discovered that this is achieved by providing an intravenously administrable liquid composition characterized by having a pH of .

[0010] The non-chemically modified complete molecular type γ-globulin of the present invention is: (1) It is in its natural state and has not undergone any modification or change, and therefore it is a fragment of γ-globulin, Fab, F(ab')2. , does not include FC, etc.
There is little decrease in antibody titer, and at the same time, there is no decrease in antibody spectrum.■ Anti-complement action (complement fixation) is 20 units (CH50 value), which is considered safe according to Japanese biological product standards.
It is said to meet the conditions that it is sufficiently lower than .

The non-chemically modified complete molecular type γ-globulin used in the present invention may be obtained by any method as long as it is in its natural state and has a low anti-complement value. In particular, the most efficient method is one that can be produced using existing equipment and is obtained by acidifying γ-globulin for intramuscular injection, which has already been used as a medicine, to separate its aggregates. However, if manufacturing complexity and yield reduction are not a problem, treatment with a nonionic surfactant can remove γ-globulin aggregates that cause anti-complement effect, and γ-globulin aggregates with low anti-complement value can be - It is preferable to use globulin. Thus, in the present invention, it is preferred that the monomer concentration of the non-chemically modified fully molecular γ-globulin is greater than 95%.

[0012] Such non-chemically modified complete molecular type γ-globulin is produced, for example, as follows.

(Raw material) As a starting material, a fraction containing immunoglobulin is used, and this is not particularly limited as long as it is derived from human plasma and contains an immunoglobulin fraction. Specifically, pastes of fractions II+III, fraction II, and equivalent fractions containing immunoglobulins obtained by ethanol fractionation of Cohn may be mentioned. The starting material may also include human blood group antibodies, kallikrein, prekallikrein, IgM, IgG polymers, and the like.

(Production method) (1) Polyethylene glycol (PEG) treatment A fraction containing γ-globulin, which is a starting material, is treated with low concentration PEG, and the supernatant is collected.

First, starting materials are suspended in a suitable aqueous solvent. As a solute in an aqueous solvent, e.g. sodium chloride,
Sodium phosphate, potassium phosphate, acetic acid, sodium acetate, citric acid, sodium citrate, etc. may be included.

[0016] This suspension has a molecular weight of 1,000 to 10,0
00 (preferably about 2,000-6,000) PEG (eg, by mixing both). The processing conditions include a protein concentration of 1 to 20 w/v% (especially 5 to 15 w/v%).
), PEG concentration 4-10 w/v% (especially 4-8 w/v%
), pH 4-6 (especially 4.5-5.5), ionic strength 0
．． 0001~0.1M (especially 0.0001~0.01M
) is preferable.

[0017] The treatment is carried out by adding PEG to the γ-globulin-containing fraction and stirring the mixture usually at about 0 to 4°C for 30 minutes to 6 hours.

[0018] Then, for example, centrifugation (6000-8000
00 rpm for 10-30 minutes) and collect the supernatant.

Furthermore, this supernatant is treated with high concentration PEG and the precipitate is collected.

That is, the above supernatant has a molecular weight of 1,000 to 10
,000 (preferably 2,000-6,000) PEG
further processing (for example, mixing both). The processing conditions include a protein concentration of 1 to 20 w/v% (especially 5
-15 w/v%), PEG concentration 10-15 w/v% (especially 11-13 w/v%), pH 6-9 (especially 7.5-8
．． 5), ionic strength 0.0001 to 0.1M (especially 0.
0001 to 0.01M). The treatment is usually performed by stirring at about 0 to 4°C for 30 minutes to 6 hours.

[0021] After that, for example, centrifugation (6000-8000
00 rpm for 10-30 minutes) to collect the precipitate.

■ Anion exchanger treatment This is an operation in which the γ-globulin-containing fraction is dissolved in an aqueous solvent and then contacted with an anion exchanger to recover the non-adsorbed fraction.
This is done to remove G polymer.

(i) Anion exchanger An anion exchanger has an anion exchange group bound to an insoluble carrier, and the anion exchange groups include diethylaminoethyl (DEAE) group and quaternary aminoethyl (QAE) group.
groups, etc., and insoluble carriers such as agarose, cellulose,
Examples include dextran and polyacrylamide.

(ii) Treatment method The γ-globulin-containing fraction is dissolved in a suitable aqueous solvent. The aqueous solvent is an aqueous solution having a pH of 4 to 7, preferably 5 to 6, a low ionic strength, preferably 0.0001 to 0.1M, and may contain the same solute as in the PEG treatment in (2) above. The protein concentration is 1 to 15 w/v% (especially 3 to 10
w/v%) is preferred.

Further, contact treatment is carried out with the anion exchanger equilibrated with the above aqueous solvent. For the treatment, either a batch method or a column method may be used.

For example, in the batch method, the two are mixed at a ratio of about 10 to 100 ml of γ-globulin solution to 1 ml of anion exchanger, stirred at 0 to 4°C for about 30 minutes to 2 hours, and then centrifuged. (6000~8000rpm,
10-30 minutes) and collect the supernatant.

In the column method, 1 ml of anion exchanger is brought into contact with the γ-globulin solution at a ratio of about 10 to 100 ml, and the non-adsorbed fraction is collected.

■ Treatment γ with immobilized diamino compound
- An operation in which a globulin-containing fraction is contacted with an immobilized diamino compound to recover a non-adsorbed fraction, and is performed to remove prekallikrein or kallikrein.

(i) Preparation of immobilized diamino compound The immobilized diamino compound is a diamino compound immobilized on an insoluble carrier.

As the diamino compound, aminobenzamidine, aminobenzguanidine, lysine, arginine, etc. can be used.

On the other hand, examples of insoluble carriers include agarose,
Cellulose, dextran, silica gel, glass, etc. are used.

[0032] Immobilization may be carried out according to known methods. For example, agarose, cellulose, etc., for example, CNBr
A diamino compound can be immobilized by an activation method, or for silica gel, glass, etc. by an oxirane method.

(ii) Processing method The γ-globulin-containing fraction is treated at a protein concentration of 1 to 15 w/v%.
(especially 3 to 10 w/v%) and pH 5 to 8 (especially p
H6-7), contact treatment with the immobilized diamino compound under conditions of ionic strength of 0.0001 to 0.1M (particularly 0.0001 to 0.01M). In this case, both the batch method and the column method are preferably used.

For example, in the batch method, the fractions are mixed at a ratio of about 10 to 100 ml to 1 ml of the immobilized diamino compound, and heated at 0 to 10°C, preferably 0 to 4°C for 30 minutes.
After stirring for 0 minutes to 4 hours, preferably 40 minutes to 2 hours, centrifugation (6000 to 8000 rpm, 10 to 30 minutes)
(min) and collect the supernatant.

[0035] Even with the column method, 1 ml of immobilized diamine compound
The non-adsorbed fractions are collected by contacting the two at a ratio of about 10 to 100 ml with each other.

■ Immobilized Human Blood Group Substance Treatment This is an operation in which the γ-globulin-containing fraction is contacted with an immobilized human blood group substance and the non-adsorbed fraction is collected, and is carried out to remove human blood group antibodies. be exposed.

(i) Preparation of immobilized human blood group substance The immobilized human blood group substance is a human blood group substance immobilized on an insoluble carrier.

Human blood group substances may be prepared using known methods. For example, after hemolyzing or sonicating human A, B, AB or O red blood cells in a hypotonic solution,
It can be obtained by purification by ammonium sulfate fractionation method or PEG fractionation method. In addition, Synsolve A, Synsolve B (C
An artificially synthesized blood type substance such as hembiomed (manufactured by LTD.) may also be used.

Furthermore, this human blood group substance is said to be effective in inactivating contaminating viruses after being dissolved in physiological saline, for example, at about 50 to 70°C, preferably at about 60°C.
for 7 to 13 hours, preferably about 10 hours, or about 8
about 1 to 40 at 0 to 130°C, preferably 95 to 121°C
Heat treatment is performed for 2 to 30 minutes, preferably 2 to 30 minutes. after that,
Centrifuge to remove unnecessary substances and dialyze against distilled water to obtain each human blood group substance.

On the other hand, examples of insoluble carriers include agarose,
Cellulose, dextran, silica gel, glass, etc. are used.

[0041] Immobilization may be carried out according to known methods. For example, human blood group substances can be immobilized on agarose, cellulose, etc. by the CNBr activation method, and on silica gel, glass, etc., by the oxirane method.

(ii) Processing method The γ-globulin-containing fraction is treated at a protein concentration of 1 to 15 w/v%.
(especially 3-10 w/v%) and pH 5-8 (especially 6-8).
7), ionic strength 0.0001-0.1M (especially 0.0
001 to 0.01 M), and is contacted with the immobilized human blood group substance equilibrated with the above aqueous solvent. At that time, either a batch method or a column method may be used.

For example, in the batch method, 1 ml of the immobilized human blood group substance is mixed with about 10 to 100 ml of solution, and the mixture is heated at 0 to 10°C, preferably 0 to 4°C, for 30 minutes to 4 hours, preferably 30 minutes. After stirring for about 2 hours, centrifugation (6000-8000 rpm, 10-30 minutes) is performed to collect the supernatant.

[0044] Even with the column method, 1 ml of immobilized human blood group substance
The two are brought into contact with each other at a ratio of about 10 to 100 ml of the solution to be treated, and the non-adsorbed fraction is collected.

■ Heat treatment In the presence of a stabilizer, the decrease in antibody activity of immunoglobulin is kept to a minimum, but contaminating HB virus and AIDS
Viruses, etc. are heat-treated under conditions to completely inactivate them. The heat treatment is performed in a dry state with a humidity content of 3% or less (ie, dry heat treatment) or in a solution state, that is, an aqueous solution of immunoglobulin (ie, liquid heat treatment).

Stabilizers used during heat treatment include disaccharides (eg, saccharose, maltose), sugar alcohols (eg, sorbitol, mannitol), and
is suitably exemplified.

The amount of stabilizer added is 0.5 to 5 w/v% (preferably 1 to 3 w/v%) of disaccharides, sugar alcohols, etc. in the dry heat treatment method, and 0.5 to 5 w/v% (preferably 1 to 3 w/v%) of disaccharides, sugar alcohols, etc. in the liquid heat treatment method. Sugar alcohol etc. at 10w/v% or more (preferably 10-40%)
w/v%) is preferably used.

[0048] The amount of immunoglobulin to be heated is:
In the dry heat treatment method, it is suitable to adjust the protein content to 1 to 10 w/v% (preferably 3 to 7 w/v%). In the liquid heat treatment method, the protein amount is 0.1 to 3
It is preferable to adjust it to 0 w/v% (preferably 5 to 20 w/v%).

In the case of dry heat treatment, the heat treatment is performed after adding a stabilizer, followed by sterilization filtration if necessary, and the water content is reduced to 3% or less, preferably 1% or less, by, for example, freeze-drying. The conditions for freeze-drying are under a vacuum of 0.5 mmHg,
An example is 24 to 96 hours at 40°C. Then, for example, 50 to 70°C (preferably about 60°C), 10 to 70°C
The treatment is carried out for 200 hours (preferably about 50 to 100 hours).

Furthermore, by carrying out this heat treatment step under an inert gas atmosphere, stability during heating can be further improved. Examples of the inert gas include nitrogen gas, argon, and helium.

In the case of liquid heat treatment, the pH of the aqueous solution is set to 4.
5 to 6.5, preferably pH 5 to 6, and in the liquid heat treatment method, the treatment is performed at, for example, 50 to 70°C (preferably about 60°C) for 10 minutes to 20 hours (preferably about 10 hours).

[0052] Depending on the purpose, the above-mentioned operations can be used in appropriate combinations for the production (purification) of the γ-globulin.

[0053] As a preferable specific example, ■liquid heat treatment→
Treatment steps such as (1) PEG treatment → (2) anion exchanger treatment can be used.

(Preparation of liquid composition) The obtained non-chemically modified complete molecular type γ-globulin was prepared by a conventional method.
Prepare an aqueous solution so that the concentration is w/v% (preferably 3 to 7 w/v%), and adjust the pH to 5.5±0.2 (preferably about 5%).
．． 5), low electrical conductivity, preferably electrical conductivity of 1 mmho or less (
In particular, it is adjusted by known means so that it is 0.6 mmho or less (both calculated at 8°C), and then sterile filtration, dispensing, etc. are performed based on normal formulation techniques. In this way, a non-chemically modified complete molecular type γ-globulin liquid composition (preparation) that can be administered intravenously is prepared.

[0055] In the present invention, "substantially sorbitol-free" means that it does not contain an amount that would cause side effects such as liver and kidney damage caused by sorbitol in patients with sorbitol metabolic disorders. .

Other known additives may also be used. For example, polyethylene glycol (molecular weight 1000-1
0000) 0.1-1 w/w%, human serum albumin 0
．． Examples include 5 to 5 w/w%, mannitol 1 to 10 w/w%, and the like.

[0057]

Effect of the invention: According to the present invention, there is no increase in γ-globulin polymers not only during long-term storage but also during in vivo administration, and no increase in anti-complement value is observed, and the appearance and properties are maintained even during long-term storage. Provided is a stable liquid composition (preparation) of non-chemically modified complete molecular type γ-globulin that can maintain good sorbitol metabolism and can be administered even to patients with sorbitol metabolic disorders.

[0058]

EXAMPLES The present invention will be explained in more detail with the following examples and test examples. In addition, in the test example, the test was conducted by the following method.

(Test method) Since turbidity is a problem in appearance, O. D. Absorbance at 600 nm was measured.

[0060] The polymer was quantitatively analyzed by high performance liquid chromatography.

[0061] The anti-complement level was measured by the method of Kabat and Mayer [Experimental immunity].
mistry, 225 (1961)] and Nishioka,
The method of Okada [Biochemistry of Immunology, 103, 1972 (Kyoritsu Shuppan)] was followed. That is, the number of units reduced by adding the sample from 100 units of complement was measured, and the unit of reduction was expressed as the anti-complement value.

[0062] Measles antibody titer was determined by Hemagglutinat.
It was measured by the ion Inhibition Test method and expressed in international units (IU/100mg).

Test Example 1 Liquid compositions prepared under various conditions using non-chemically modified complete molecular type γ-globulin were stored at 25° C. for 3 months, and their stability was investigated.

(1) pH γ-globulin concentration 5 w/v%, electrical conductivity 1 mmho (8
℃ conversion), the liquid composition adjusted to various pH was heated at 25℃ for 3
After storage for a month, it was returned to neutrality and used for testing. The results are in Table 1
As shown in , it was particularly stable at about pH 5.5.

[0065]

[Table 1]

(2) Conductivity Liquid compositions having a γ-globulin concentration of 5 w/v%, a pH of 5.5, and various conductivities were stored at 25° C. for 3 months and then subjected to testing. The results are shown in Table 2, and it was particularly stable when the conductivity was 1 mmho or less.

[0067]

[Table 2]

Test Example 2 A liquid composition containing 5 w/v % of non-chemically modified complete molecular type γ-globulin, an electrical conductivity of 1 mmho (calculated at 8° C.), and a pH of 5.5 was prepared. This composition had the following properties at the time of preparation. Appearance properties
: Colorless transparent polymer (%) : 0.
00 Anti-complement titer (CH50/ml): 8 Measles antibody titer (IU): 32

The storage stability of this composition was investigated. The results are shown in Table 3.

[0070]

[Table 3]

Example 1 1 kg of corn fraction II+III paste was added to 1 kg of distilled water.
After suspending in L and adjusting the pH to 5.5, centrifugation was performed, the supernatant was collected, 50 g of sorbitol (final concentration 33 w/v%) was added per 100 ml of supernatant, and the suspension was suspended at 60°C for 1
Heat treatment was performed for 0 hours.

After the heat treatment and adjusting the pH to 5.5,
Add PEG #4000 to a final concentration of 6%,
Centrifugation was performed at 2°C.

[0073] After adjusting the pH of the obtained supernatant to 8.0 using 1N sodium hydroxide, PEG #4000 was added to a final concentration of 12%, centrifugation was performed at 2°C, and the precipitated fraction was An IgG fraction was obtained.

This fraction was dissolved in distilled water, and DEAE-Sephadex was added to 100 ml of this IgG solution (50 ml).
1 ml per ml solution), contact treatment was carried out for about 1 hour under conditions of 0 to 4° C., and the supernatant (IgG solution) was collected by ultracentrifugation (7000 rpm, about 20 minutes).

[0075] This IgG solution was adjusted to 5% IgG solution with distilled water, the pH of the solution was adjusted to 5.5 with sodium acetate, and the sorbitol used in the previous step was replaced with UF membrane (manufactured by Amicon)
It was removed using

This aqueous solution (conductivity: about 1 mmho) was sterilized and filtered to prepare a liquid immunoglobulin preparation for intravenous injection.

Example 2 To the IgG solution obtained in Example 1, polyethylene glycol #4000 was added to the final concentration of 0.5%, human serum albumin to 1%, and D-mannitol to 2%. The product was similarly sterilized and filtered to prepare a liquid immunoglobulin preparation for intravenous injection.

Claims (4)

[Claims] 1. A low conductivity solution containing non-chemically modified complete molecular γ-globulin, which is characterized by being substantially free of sorbitol and having a pH of about 5.5±0.2. A liquid composition that can be administered intravenously. 2. The composition of claim 1, wherein the monomer content of the non-chemically modified fully molecular γ-globulin is greater than 95%. 3. The composition according to claim 1, which has an electrical conductivity of 1 mmho (calculated at 8° C.) or less. 4. The composition according to claim 1, which contains at least one stabilizer selected from polyethylene glycol, human serum albumin, and D-mannitol.